Microsomal Cytochrome P450 Monooxygenase System Research Articles

Biosensor selection of small compounds, modulating a complex formation between cytochrome P450s and NADPH-dependent P450 oxidoreductase

The study of the effect of low-molecular-weight compounds (substrates, endogenous metabolites, drugs and xenobiotics) on the kinetic and equilibrium parameters of functionally significant binary protein-protein interactions (PPIs) is of both fundamental and clinical importance. The surface plasmon resonance (SPR) is the method of the first choice for studying PPIs. Earlier, SPR analysis revealed the modulating effect of steroidal substrates on the affinity of interactions between steroidogenic microsomal cytochromes P450 (CYP) and their redox partner (cytochrome b5). In this work, we have shown the suitability of the experimental approach for assessing the selective effect of the cofactor NADPH on the interaction between cytochromes CYP3A4 or CYP2E1 with NADPH-dependent P450 oxidoreductase (CPR). Experiments have shown that the CYP3A4/CPR complex is not modulated by NADPH, while the dissociation rate of the CYP2E1/CPR complex in the presence of NADPH significantly decreased: the koff values in the absence and presence of NADPH were (3.6 ± 0.2) • 10-3 s-1 and (3.8 ± 0.2) • 10-4 s-1, respectively. Thus, in the presence of NADPH, an increase in the affinity of CYP2E1/CPR complex formation by approximately one order of magnitude was observed, while NADPH did not affect the kon value of this complex. Co-injection of NADPH at the CYP2E1/CPR complex preformed in the absence of NADPH had minor influence on the koff values (<10%). This suggests a stabilizing role of NADPH for the CYP2E1/CPR complex formation. Thus, the use of our approach made it possible to assess the effect of the main electron supplier for the microsomal cytochrome P450 monooxygenase system on the kinetic rate constants of CYP/CPR complexes.

Structural and Functional Studies of the Membrane-Binding Domain of NADPH-Cytochrome P450 Oxidoreductase.

NADPH-cytochrome P450 oxidoreductase (CYPOR), the essential flavoprotein of the microsomal cytochrome P450 monooxygenase system, is anchored in the phospholipid bilayer by its amino-terminal membrane-binding domain (MBD), which is necessary for efficient electron transfer to cytochrome P450. Although crystallographic and kinetic studies have established the structure of the soluble catalytic domain and the role of conformational motions in the control of electron transfer, the role of the MBD is largely unknown. We examined the role of the MBD in P450 catalysis through studies of amino-terminal deletion mutants and site-directed spin labeling. We show that the MBD spans the membrane and present a model for the orientation of CYPOR on the membrane capable of forming a complex with cytochrome P450. EPR power saturation measurements of CYPOR mutants in liposomes containing a lipid/Ni(II) chelate identified a region of the soluble domain interacting with the membrane. The deletion of more than 29 residues from the N-terminus of CYPOR decreases cytochrome P450 activity concomitant with alterations in electrophoretic mobility and an increased resistance to protease digestion. The altered kinetic properties of these mutants are consistent with electron transfer through random collisions rather than via formation of a stable CYPOR-P450 complex. Purified MBD binds weakly to cytochrome P450, suggesting that other interactions are also required for CYPOR-P450 complex formation. We propose that the MBD and flexible tether region of CYPOR, residues 51-63, play an important role in facilitating the movement of the soluble domain relative to the membrane and in promoting multiple orientations that permit specific interactions of CYPOR with its varied partners.

Reponses of the Hepatic Microsomal Cytochrome P450 Monooxygenase System in Rock Bream Oplegnathus fasciatus Exposed to Tributyltin (TBT)

Abstract The study was conducted to investigate the responses of the hepatic microsomal cytochrome P450 monooxygenase system in the rock bream Oplegnathus fasciatus after chronic exposure to 0, 1, 2, 4, and 8 μg/L tributyltin (TBT) concentrations for 4 weeks. Hepatic cytochrome 450 content and ethoxyresorufin O -deethylation (EROD) activity were found to significantly increase in fish treated with the higher concentration of TBT (≥4 μg/L); however, no significant changes were observed in penthoxyresorufin O -deethylation (PROD) activity in all treated groups compared to the control group. These findings suggest that exposure to a low TBT concentration (≥4 μg/L) has the potential to induce cytochrome 450 content and EROD enzyme activity in hepatic tissue in the rock bream. Key words: Oplegnathus fasciatus , Tributyltin, Cytochrome 450, EROD, PROD Introduction Many aquatic ecosystems are faced with spatially or tem-porally alarmingly high levels of xenobiotic chemicals (Brack et al., 2002; Diez et al., 2002). Tributyltin (TBT) is a typi-cal synthetic pollutant, which was selected for investigation in this study. TBT is an organotin compound used primarily as a biocide in antifouling paints for ships, boats, and fishing nets. The use of TBT as an antifouling agent in paint has been banned for small boats and fishing nets in most countries as it has been shown to be toxic to aquatic life and is an endo-crine disrupting chemical that generates severe reproductive effects in aquatic animals (Horiguchi et al., 1994). Reproduc-tive impairment in gastropods has been reported at low TBT concentrations (Gibbs et al., 1988). TBT has been shown to induce imposex and intersex in brosobranchs (Oehlmann et al., 1998), and impaired reproductive function in fish has also been described (Mc Allister and Kime, 2003).Some studies have reported toxic effects of TBT such as morphological and functional alterations of teleost gills in aquatic media (Byrne et al., 1989; Schwaiger et al., 1992; Tsu -da et al., 1992; Wang and Huang, 1998). TBT has also been shown to markedly inhibit fish hepatic CYP and in particu-lar the isozyme cytochrome P4501A1 (CYP1A), both in vivo and vitro (Fent and Bucheli, 1994; Morcillo and Porte, 1997). The CYP1A isozyme plays a key role in the biotransformation of xenobiotic compounds, leading to either their detoxifica-tion or bioactivation, while other CYP forms are important

Cross-Linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b5

Cytochrome b(5) (cyt b(5)) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b(5) modulation of CYP catalysis. Both cyt b(5) and apo b(5) are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b(5) with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b(5) with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b(5) and apo b(5) were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B' loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b(5) binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b(5) and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b(5) can dock with CYP3A4 in a manner analogous to that of holo b(5), so electron transfer from cyt b(5) is not required for its effects.

Involvement of cytochrome P450 in hydroxylation of propylbenzene by Fusarium moniliforme strain MS31

Fusarium moniliforme strain MS31 can oxidize propylbenzene to ( R)-1-phenylpropanol with what may be a cytochrome P450. Hydroxylation of propylbenzene needed molecular oxygen, and NADPH as a coenzyme gave a higher yield than NADH. The hydroxylation proceeded further when FAD and FMN were added than in their absence, suggesting that the enzyme was a flavo-protein. Carbon monoxide inhibited the hydroxylation, as did other cytochrome P450 inhibitors such as SKF 525A and miconazole. These characteristics matched those of a microsomal cytochrome P450 monooxygenase system that contained NADPH-cytochrome P450 reductase.

Involvement of Cytochrome P450 in Hydroxylation of Propylbenzene by Fusarium moniliforme Strain MS31

Fusarium moniliforme strain MS31 can oxidize propylbenzene to (R)-1-phenylpropanol with what may be a cytochrome P450. Hydroxylation of propylbenzene needed molecular oxygen, and NADPH as a coenzyme gave a higher yield than NADH. The hydroxylation proceeded further when FAD and FMN were added than in their absence, suggesting that the enzyme was a flavo-protein. Carbon monoxide inhibited the hydroxylation, as did other cytochrome P450 inhibitors such as SKF 525A and miconazole. These characteristics matched those of a microsomal cytochrome P450 monooxygenase system that contained NADPH-cytochrome P450 reductase.

Microsomal cytochrome P-450 monooxygenase system and its drug-metabolizing activity after partial portal vein ligation in the Rat.

Percutaneous transhepatic portal vein embolization (PTPE) has been used to decrease the risk of hepatic failure after hepatectomy in patients with poor liver function. The effect of PTPE on hepatic drug-metabolizing activities is not clear. Therefore we examined the effect of portal vein branch ligation, a model of PTPE, on hepatic drug-metabolizing activities in Sprague-Dawley rats. Ligated and nonligated lobes were harvested separately. Drug-metabolizing activities and concentrations of components of the microsomal cytochrome P-450 monooxygenase system were examined. In ligated lobes, drug-metabolizing activities (lidocaine and aminopyrine) and enzymatic concentrations of the microsomal cytochrome P-450 monooxygenase system gradually decreased over 10 days. In nonligated lobes these functions were depressed rapidly to 60% of those before PBL but then recovered 10 days after PBL. From the viewpoint of drug metabolism, hepatic dysfunction occurred in both ligated and nonligated lobes.

Interaction of tributyl- and triphenyltin with the microsomal monooxygenase system of molluscs and fish from the Western Mediterranean

The in vitro interaction of tributyltin (TBT) and triphenyltin (TPhT) with the microsomal cytochrome P450 monooxygenase system of different marine organisms has been evaluated. The selected organisms were two bivalve species, the mussel Mytilus galloprovincialis and the clam Tapes decussata, and the gastropod Thais haemastoma. The fish Mullus barbatus was also studied and the results compared with those obtained for invertebrates. The microsomal fraction isolated from the hepatopancreas or liver of these organisms was incubated in the presence of different concentrations of TBT and TPhT and the interaction with cytochrome P450, cytochrome b 5, NAD(P)H cytochrome c reductases and 7-ethoxyresorufin O-deethylase (EROD) activity evaluated. Direct effects on spectrally determined P450 and b 5 were only studied in mussels and detected at very high concentrations of TBT or TPhT (1 mM). In general, TBT and TPhT differed in their specificity. TBT led to inhibition of both NADH and NADPH cytochrome c reductase activity in the studied molluscs, whereas TPhT showed little effect. On the contrary, no significant effects on fish hepatic NAD(P)H cytochrome c reductases were detected. EROD activity, however, was significantly inhibited by both TBT and TPhT in Mullus barbatus at a concentration as low as 5–10 μM. The study shows speciesrelated differences and selective effects of TBT and TPhT on different components of the microsomal monooxygenase system.

40] Crystallization studies of NADPH-cytochrome P450 reductase

This chapter focuses on the crystallization studies of NADPH-cytochrome P450 reductase (CPR). NADPH-cytochrome P450 reductase is an essential component of the microsomal cytochrome P450 monooxygenase system. It is an integral membrane protein that catalyzes the transfer of electrons from NADPH to cytochrome P450 in the oxidative metabolism of the endogenous and exogenous substrates. The three-dimensional structure of CPR by X-ray analysis can reveal, at the molecular level, the interactions among the pyridine nucleotide NADPH, the flavins, and the polypeptide chain of CPR, and the spatial relationship among the different domains of CPR that allow for efficient electron transfer. In addition, the nature of the binding site for cytochrome P450 can be identified, providing the structural basis for the ability of CPR to interact with scores of different cytochromes P450. Obtaining crystals of CPR establishes the basis for the full structural analysis of CPR by X-ray diffraction methods. The crystallization conditions obtained from the CPR studies can be used as a starting point for crystallization of other FMN- and FAD-containing proteins, such as nitric oxide synthase.

Biphasic response for hepatic microsomal enzyme induction by 2,3,7,8-tetrachlorodibenzo- p-dioxin in C57BL/6J and DBA/2J mice

The induction of the murine hepatic microsomal cytochrome P-450 monooxygenase system by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) was studied over a wide range of doses, including those associated with acute toxicity. Studies were conducted in two inbred strains of mice which vary at the Ah receptor and at a number of other genetic loci. C57BL/6J mice possess at high-affinity Ah receptor and are responsive to enzyme inductive effects of TCDD, whereas DBA/2J mice do not possess a high-affinity receptor and are less responsive to TCDD. In a dose-response study, 7-ethoxyresorufin O-deethylase (EROD) activity appeared to be maximally induced in C57BL/6J and DBA/2J mice at 7 days following exposure to 3 and 30 μg of TCDD/kg respectively. Very similar results were reported previously for the induction of aryl hydrocarbon hydroxylase activity in these strains of mice. However, at higher doses of TCDD (at least 45 μg/kg for C57BL/6J and 300 μg/kg for DBA/2J), EROD activity was further increased (2-fold) from the apparent maximal (plateau) level, resulting in an unusual biphasic log dose-response relationship. EROD activity remained at these elevated rates in both strains for doses approaching and exceeding the respective ld 50 values for each strain. To further characterize this biphasic induction phenomenon, cytochrome P-450 content, benzo[ a]pyrene metabolism, and EROD and NADPH-cytochrome P-450 reductase activities were measured 1, 3 and 7 days after TCDD administration to C57BL/6J (3 and 150 μg/kg) and DBA/2J (30 and 600 μg/kg) mice. Maximal responses occurred in both strains at 3 days for all doses. In both strains, TCDD produced a dose-dependent increase in cytochrome P-450 content, EROD, and benzo[ a]pyrene metabolism. Furthermore, a 2-fold induction of reductase activity was observed in each strain following exposure to the respective high doses. Induction of cytochrome P 1-450 and P 3-450 was also measured by Western immunoblot, using antisera raised against the homologous rat isozymes. In both strains, TCDD produced a dose-related increase in two protein-staining bands recognized by anti-P-450 BNF-B (P 1-450) and anti-P-450 BNF/ISF-G (P 3-450) respectively. The extended induction of hepatic microsomal monooxygenase activities at the respective high doses of TCDD appears to be due, in part, to increases in NADPH-cytochrome P-450 reductase activity and cytochromes P 1-450 and P 3-450 content. Significant alterations in the expression of the cytochrome P-450 monooxygenase system following exposure to high doses of TCDD may be associated, in part, with the delayed acute toxicity reported at this level of exposure.

Microsomal cytochrome P450 monooxygenases in the house fly ( Musca domestica L.): Biochemical changes associated with pyrethroid resistance and phenobarbital induction

Differences in the microsomal cytochrome P450 monooxygenase system and its inducibility by sodium phenobarbital (PB) were investigated in insecticide-susceptible (S+) and pyrethroid-resistant (LPR) house fly strains. In comparison to the S+ strain, LPR had higher levels of total cytochrome P450s, cytochrome b 5, NADPH-cytochrome c (P450) reductase, and six monooxygenase activities: aryl hydrocarbon hydroxylation (AHH), aldrin epoxidation (AE), 7-ethoxycoumarin O-deethylation (ECOD), p-chloro- N-methylaniline N-demethylation (PCMA), ethoxyresorufin O-deethylation (EROD), and methoxyresorufin O-demethylation (MROD). The largest differences between strains were noted for MROD (64×) and EROD (56×) activities, and these appeared to be correlated with the monooxygenase-mediated pyrethroid resistance in the LPR strain. Comparisons between susceptible and resistant strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the LPR strain showed a marked increase in the 54,400-Da protein band, suggesting this may be a cytochrome P450 isozyme involved in conferring part of the monooxygenase-mediated pyrethroid resistance. In vivo administration of 0.25% PB in sugar water to S+ flies induced higher levels of three components of the microsomal monooxygenase system, as well as increased levels of all six monooxygenase activities. In contrast, PB treatment had no significant effect on the level of cytochrome P450 or on EROD, AHH, or AE activity in LPR flies. Additionally, PB caused a decrease in the level of cytochrome b 5 and MROD and PCMA activities, while ECOD activity and the level of NADPH-cytochrome c (P450) reductase were significantly increased. SDS-PAGE indicated that induction of S+ flies with PB caused an increase in at least seven proteins with apparent molecular masses of 47,400; 49,400; 51,500; 52,200; 54,400; 56,000; and 57,500 Da. However, no observable difference was noted in PB-induced LPR flies. Differences in monooxygenase activities, in conjunction with the changes in turnover numbers of ECOD and AE activities in S+ flies and ECOD and MROD activities in LPR flies, suggest that PB treatment induced both quantitative and qualitative changes in the S+ and LPR cytochrome P450 monooxygenases.

Partial purification of pisatin demethylase, a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and ω-aminohexyl-agarose. The reductase was further purified by chromatography on 2′,5′-ADP-agarose to a specific activity of about 16 μmoles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after ω-aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.

Oxidase and oxygenase function of the microsomal cytochrome P450 monooxygenase system.

The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital-pretreated rats. Correcting a quenching of O2(-) radicals by microsomes, a stoichiometry of O2(-) to H2O2 close to 2:1 was obtained. This, and the fact that pseudo-substrates of microsomal cytochrome P450 like perfluoro-n-hexane and perfluorinated cyclohexane did not increase H2O2 formation in a catalase-inhibited assay, rules out a two-electron reduced oxygen species as the source of H2O2. The rates of O2(-) as well as H2O2 generation in the presence of 7-ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H2O2 formation dropped to 55%. It was concluded that most of the O2(-) originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H2O2 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton-assisted decomposition of oxy-cytochrome P450 appears likely. H2O2 formation was only slightly decreased at 20 microM dioxygen suggesting that H2O2 formation via cytochrome P450 should also occur in vivo.

The induction of cytochrome P-448 dependent benzo(a)pyrene hydroxylase in [formula omitted

When grown in high concentrations of glucose, the yeast Saccharomyces cerevisiae produces a microsomal cytochrome P-450 monooxygenase system which is capable of hydroxylating benzo(a)pyrene. The addition of benzo(a)pyrene to the yeast during growth causes only a small increase in cytochrome P-448 levels but results in a dramatic improvement in the apparent kinetics of benzo(a)pyrene hydroxylation as measured by a decrease in the Michaelis constant and an increase in maximal velocity. Dimethylnitrosamine, phenobarbital and 3-methylcholanthrene also induce this enzyme to various degrees. Yeast pretreatment with β-naphthoflavone did not affect this enzyme, yet pretreatment with lanosterol resulted in a decreased affinity for benzo(a)pyrene. The addition of benzo(a)pyrene to yeast growing at low glucose concentration does not induce cytochrome P-448. The implications of these findings with regard to the presence of multiple forms of cytochromes P-448 P-450 in yeast are briefly discussed.

Interactions of prostaglandins with hepatic microsomal cytochrome P-450

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGAI, PGA2, PGE 1 , PGE 2 , PGF 1α and PGF 2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K s ) were highest with PGA 1 and PGA 2 . With liver microsomes from control or 3-methylcholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a weak spectrum, designated here as type “II” was at times observed. With microsomes from phenobarbital (Pb)-treated rats only PGA 1 and PGA 2 yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA 1 and PGA 2 to liver microsomes from Pb-treated rats inhibited the microsomal mediated hydroxylation of hexobarbital. The inhibition by PGA 1 was competitive; the K i = 8.2 × 10 −4 M was found to be similar in magnitude to the K s = 7.3 × 10 −4 M of PGA 1 observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.

Interactions of prostaglandins with hepatic microsomal cytochrome P-450

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGAI, PGA2, PGE 1, PGE 2, PGF 1α and PGF 2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K s) were highest with PGA 1 and PGA 2. With liver microsomes from control or 3-methylcholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a weak spectrum, designated here as type “II” was at times observed. With microsomes from phenobarbital (Pb)-treated rats only PGA 1 and PGA 2 yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA 1 and PGA 2 to liver microsomes from Pb-treated rats inhibited the microsomal mediated hydroxylation of hexobarbital. The inhibition by PGA 1 was competitive; the K i = 8.2 × 10 −4 M was found to be similar in magnitude to the K s = 7.3 × 10 −4 M of PGA 1 observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.